4-Hydroxy-nonenal (4-HNE) and related aldehydes are established as important agents formed in the course of the lipid peroxidation reactions of oxidative stress. Their biological activities are associated with their chemical reactivities, resulting in their adduction to proteins, other lipids and DNA. The immediate cytotoxic actions include changes in gene expression, growth regulation and the induction of apoptosis, while the longer term effects are the accumulation of damaged cellular components in degenerative diseases and aging, and the potential for mutagenic changes through binding to DNA. The mechanism of biosynthesis of 4-HNE and related aldehydes is not understood and is a major focus of this proposal. We have evidence for two main pathways to 4-HNE in both of which 4-hydroperoxy-nonenal is the immediate precursor. One pathway converts fatty acid hydroperoxides with an omega-6 hydroperoxide (13-hydroperoxylinoleate or 15-hydroperoxyeicosatetraenoate (15-HPETE)), and a second involves 9-hydroperoxylinoleate and l l-HPETE. The proposed mechanisms will be examined utilizing identification of intermediates by HPLC, UV, mass spectrometry, and NMR. The Specific Aims are to investigate: Mechanisms of formation from 13-hydroperoxylinoleic acid and 15-HPETE Mechanisms of formation from 9-hydroperoxylinoleic acid and 11-HPETE Catalysis of 4-HNE and related aldehyde formation by cytochrome P450s Regulation of 4-HNE production by (alpha-tocopherol and molecular oxygen Evaluation of the pathways to 4-HNE in biological systems, and the biological activity of the novel intermediates in comparison to 4-HNE Elucidation of the mechanism of formation of these reactive aldehydes has practical applications in defining the parameters that influence the production of aldehydes, identification of novel and reactive chemical species involved in the synthetic pathways, in providing a perspective on the yield of products through an appreciation of the synthetic pathways, and improving the assessment of peroxide tone/oxidative stress in tissues, again through an appreciation of the mechanism of synthesis.